Compositions and methods for treating lung injuries associated with sars-cov-2 infections

ABSTRACT

The present invention provides methods and compositions for treating and preventing lung injuries due to or associated with coronavirus infections that cause Severe Acute Respiratory Syndrome, including COVID-19. More specifically the present invention provides methods for treating or preventing the lung injuries associated with SARS-CoV-2 infections, such as acute lung injury (ALI), lung fibrosis, and acute respiratory distress syndrome (ARDS). The methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a protein kinase inhibitor compound having MAP3K2/MAP3K3 inhibition activity, such as pazopanib or nintedanib, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, to a patient in need thereof. The present invention also provides devices for administering the compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/325,968 filed May 20, 2021, which claims the benefit of the filingdate of U.S. Provisional Application No. 63/029,375, filed May 22, 2020,all of which are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention provides methods and compositions for treating andpreventing lung injuries due to or associated with coronavirusinfections that cause Severe Acute Respiratory Syndrome, includingCOVID-19. More specifically the present invention provides methods fortreating or preventing the lung injuries associated with SARS-CoV-2infections, such as acute lung injury (ALI), lung fibrosis, and acuterespiratory distress syndrome (ARDS). The methods comprise administeringa therapeutically effective amount of a pharmaceutical compositioncomprising a protein kinase inhibitor compound having MAP3K2/MAP3K3inhibition activity, such as pazopanib or nintedanib, or apharmaceutically acceptable salt, solvate, or prodrug thereof, to apatient in need thereof. The present invention also provides devices foradministering the compositions.

BACKGROUND OF THE INVENTION

Coronavirus disease 2019, also known as COVID-19, is an infectiousdisease caused by the Severe Acute Respiratory Syndrome Corona Virus 2(SARS-CoV-2). The disease was first identified in 2019 in Wuhan, Hubeiprovince, China. As of early May 2020, more than 3.48 million cases havebeen reported across 187 countries and territories, resulting in morethan 246,000 deaths. See, COVID-19 Dashboard by the Center for SystemsScience and Engineering (CSSE) at Johns Hopkins University (JHU)”.ArcGIS. Johns Hopkins University. May 3, 2020.

Common symptoms of coronavirus infections include fever, cough, fatigue,shortness of breath, and loss of smell and taste. Even though themajority of cases result in mild symptoms, some progress to viralpneumonia, multi-organ failure, cytokine storm, and permanent tissue andorgan damage, such as lung damage, and death. The disease can beparticularly serious with poor outcomes for those most at risk. Some ofthe more serious risk factors include asthma, chronic lung disease,diabetes, serious heart conditions, chronic kidney disease being treatedwith dialysis, severe obesity, people aged 65 years and older, people innursing homes or long-term care facilities, and those who areimmunocompromised (such as patients undergoing cancer chemotherapy ortransplant recipients). The time from exposure to onset of symptoms istypically around five days but can range from two to fourteen days.

The disease is highly contagious. The virus is primarily spread betweenpeople during close contact, often via small droplets produced bycoughing, sneezing, or even during speaking. On surfaces, the virus canremain active for several hours or days and people can become infectedby touching a contaminated surface and then touching their face, wherethe virus can enter the body through the nose, mouth, eyes or ears. Acarrier of the disease is generally most contagious during the firstthree days of symptoms, but may spread the disease before symptomsappear or asymptomatically. The standard method of diagnosis is byreal-time reverse transcription polymerase chain reaction (rRT-PCR) froma nasopharyngeal swab.

As of May 1, 2020, no drugs or other therapeutics have been approved bythe U.S. Food and Drug Administration (FDA) to prevent or treatcoronavirus infections. Current clinical management includes infectionprevention and control measures and supportive care, includingsupplemental oxygen and mechanical ventilatory support when indicated.Treatment is managed to control symptoms and to provide palliative care.For severe cases requiring hospitalization, and in particular thoserequiring admission to an intensive care unit (ICU), mechanicalventilation may be required if blood oxygen levels become too low.However, despite the benefits of mechanical ventilation, there is therisk of ventilator-associated lung injury and pneumothorax, i.e. lungcollapse.

Two medications, hydroxychloroquine and chloroquine, which have beenused to treat malaria and autoimmune conditions such as rheumatoidarthritis and lupus have been used on an experimental basis. However, asof May 1, 2020, there has not been definitive medical data demonstratingthe effectiveness of hydroxychloroquine and chloroquine. Furthermore,the antiviral remdesivir is being explored as a treatment, based on invitro and in vivo activity in animal models against structurally similarviral pathogens. However, currently, the most currently effectivemeasures for addressing coronavirus infections are focused on diseaseprevention and spread and include hand washing, wearing of facecoverings, social distancing, and quarantining.

A consequence of coronavirus infections is the acute lung injury (ALI)and acute respiratory distress syndrome (ARDS) which can result from asevere case of infection. ALI and ARDS are the manifestations of aninflammatory response of the lung to direct or indirect insults, and arecharacterized by severe hypoxemia and a substantial reduction inpulmonary compliance due to diffuse alveolar damage, neutrophilicinflammation, and protein-rich edema in the lungs. ALI and ARDS have avery high mortality rate of about 40%. Care of these conditions islargely dependent on supportive measures and there is currently a lackof effective pharmacological interventions. Prior pharmacologicaltherapies that have been tested in patients with ALI/ARDS failed toreduce mortality. There is thus a clear unmet medical need fortherapeutic intervention of the ALI and ARDS that is often associatedwith a severe coronavirus infection.

One of the hallmarks of ALI is the abundant presence of neutrophils inthe lungs. Neutrophils are the most abundant leukocytes in humancirculation, playing important roles in innate immunity againstmicrobial infections and also contributing to inflammation-relatedtissue damage. During the inflammation, neutrophils are recruited to thesites of injury and infection from circulation through a multi-stepprocess, which includes rolling and firm adhesion on endothelial cells,intravascular crawling, diapedesis, and extravascular chemotaxis. Onceat the sites, neutrophils perform a number of tasks includingphagocytosis, release of preformed granule enzymes, and production ofreactive oxygen species (ROS). Evidence has clearly linked neutrophilsto the pathogenesis of ALI/ARDS. Although crossing of the alveolarepithelium by neutrophils does not directly cause an increase in lungepithelial permeability, neutrophils play important roles in pulmonaryedema with the underlying mechanisms that remain incompletelyunderstood.

While neutrophil extracellular traps and granule enzymes such asneutrophil elastase contribute to the pathology of ALI, including lungedema, any role of ROS in ALI/ARDS is still debatable. Neutrophilsproduce ROS primarily through the phagocyte NADPH oxidase, which is amember of the NOX family. This family consists of four cytosoliccomponents (p47^(phox), p67^(phox), p40^(phox)and Rac) and two membranesubunits (gp91^(phox)/NOX2 and p22^(phox)). When the cells are activatedby stimuli such as chemo-attractants, the cytosolic components arerecruited to the membrane components to form the active holoenzyme toproduce ROS. One of the key activation events is the phosphorylation ofthe cytosolic p47^(phox) subunit by protein kinases including PKC. Thephosphorylation disrupts auto-inhibitory intramolecular interactioninvolving the internal SH3 domains, leading to its interaction withp22^(phox) required for the activation of the NADPH oxidase. MAP3K2 andMAP3K3 are two highly conserved members of the MEK kinase (MEKK)subgroup of the MAP3K superfamily, and contain a kinase domain in the Cterminus and a PB1 domain near the N terminus. The kinase domains ofMAP3K2 and MAP3K3 share 94% sequence identity, and these two kinases areexpected to share substrates. Transient expression of the kinases invitro leads to their auto-activation and activation of ERK1 and ERK2,p38, JNK, and ERKS. In mice, these kinases are involved incardiovascular development, lymphocyte differentiation and NF-kappaBregulation. However, their roles in primary myeloid cell biology or ALIhave not been investigated. It had previously been found that theMAP3K2/MAP3K3 inhibitor, pazopanib, provides a method of treating orpreventing acute lung injury and treating or preventing lung fibrosis.See WO 2018/195084, to Wu et al., published Oct. 25, 2018.

Pazopanib is a small molecule inhibitor that was originally identifiedas inhibiting multiple protein tyrosine kinases with potentialantineoplastic activity. Pazopanib selectively inhibits vascularendothelial growth factor receptors (VEGFR)-1, -2 and -3, c-kit andplatelet derived growth factor receptor (PDGF-R), which may result ininhibition of angiogenesis in tumors in which these receptors areupregulated. It is used in the therapy of advanced renal cell carcinomaand soft tissue sarcomas. Pazopanib is known by the trade name Votrient®and is marketed by Novartis as an oral tablet formulation. Pazopanib isalso a p47^(phox) substrate-specific inhibitor of MAP3K2/MAP3K3.Pazopanib inhibits MAP3K2/MAP3K3-mediated phosphorylation of p47^(phox)at low nM levels while inhibiting other substrates of MAP3K2/MAP3K3 suchas MEK5 at μM levels. See Ibid., WO 2018/195084. In addition topazopanib, nintedanib, which is sold under Trade names OFEV and Vargatefand which is marketed by Boehinger Ingelheim also inhibitsMAP3K2/MAP3K3-mediated phosphorylation of p47^(phox) and has been shownto reduce ALI in mouse models. While pazopanib and nintedanib areinhibitors of tyrosine kinases, many other similar tyrosine kinaseinhibitors, including imatinib, do not inhibit MAP3K2/MAP3K3-mediatedphosphorylation of p47^(phox) nor reduce ALI in mouse models.

It is therefore seen there is an urgent and unmet need to treat orprevent lung injuries which can be due to or associated with infectionssuch as coronavirus. The present invention provides methods andcompositions for treating or preventing such lung injuries comprisingadministering a therapeutically effective amount of a pharmaceuticalcomposition to a subject in need thereof of a comprising a compoundcapable of inhibiting MAP3K2 and/or MAP3K3.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for treating orpreventing lung injuries due to or associated with viral infections. Themethods comprise administering a therapeutically effective amount of apharmaceutical composition to a subject in need thereof comprising aMAP3K2/MAP3K3 inhibitor, such as pazopanib or nintedanib, or apharmaceutically acceptable salt, solvate, or prodrug thereof to apatient in need thereof. More specifically, the present inventionprovides methods and compositions for treating and preventing lunginjuries associated with coronavirus infections that cause Severe AcuteRespiratory Syndrome including COVID-19.

Examples of useful compositions comprise those which can be deliveredusing an administration route such as oral, oropharyngeal, parenteral,nasal, respiratory (inhalation), intraperitoneal, intrapleural,intravenous, lanyngeal, topical, transdermal, transmucosalintratracheal, intrapulmonary, and intrabronchial. The present inventionalso provides devices for administering these compositions to thepatient.

In some embodiments the present invention provides:

A method for treating, preventing, or reducing the severity of a lunginjury associated with and/or due to a viral infection comprisingadministering a therapeutically effective amount of a pharmaceuticalcomposition comprising a compound capable of inhibiting MAP3K2/MAP3K3,or a pharmaceutically acceptable salt, ester, solvate, or prodrugthereof to a patient in need thereof.

In further embodiments, the present invention provides:

A method wherein said compound capable of inhibiting MAP3K2/MAP3K3 isselected from the group consisting of pazopanib or nintedanib, andcombinations thereof, and pharmaceutically acceptable salts, solvates,or prodrugs thereof.

A method wherein said compound capable of inhibiting MAP3K2/MAP3K3 ispazopanib, or a pharmaceutically acceptable salt, solvate, or prodrugthereof.

A method wherein the pharmaceutically acceptable salt is selected from asalt of a mineral acid selected from the group consisting ofhydrochloride, hydrobromide, hydroiodide, hydrogen sulfate, or a saltselected from mesylate, esylate, besylate, tosylate, and combinationsthereof.

A method wherein the compound capable of inhibiting MAP3K2/MAP3K3 ispazopanib hydrochloride.

A method wherein the compound capable of inhibiting MAP3K2/MAP3K3 isnintedanib, or a pharmaceutically acceptable salt, solvate, or prodrugthereof.

A method wherein the pharmaceutically acceptable salt of nintedanib isselected from a salt of a mineral acid selected from the groupconsisting of hydrochloride, hydrobromide, hydroiodide, hydrogensulfate, or a salt selected from mesylate, esylate, besylate, tosylate,and combinations thereof.

A method wherein the compound capable of inhibiting MAP3K2/MAP3K3 isnintedanib esylate.

A method wherein the compound capable of inhibiting MAP3K2/MAP3K3 isadministered using an administration route selected from the groupconsisting of oral, intra venous, parenteral, nasal, inhalation (i.e.respiratory), intratracheal, intrapulmonary, and intrabronchial.

A method wherein the compound capable of inhibiting MAP3K2/MAP3K3 isadministered using an administration route selected from the groupconsisting of nasal, inhalation (i.e. respiratory), intratracheal,intrapulmonary, and intrabronchial.

A method wherein the administration is performed using a spray device ornebulizer.

A method wherein the patient is a mammal.

A method wherein the patient is a human.

A method wherein the patient is on a ventilator (also known as arespirator or breathing machine).

A method wherein the patient is in an intensive care unit (ICU) or anemergency room (ER).

A method wherein the lung injury is selected from the group consistingof acute lung injury (ALI), lung fibrosis, and acute respiratorydistress syndrome (ARDS).

A method wherein the lung injury is acute lung injury (ALI).

A method wherein the lung injury is lung fibrosis.

A method wherein the lung injury is acute respiratory distress syndrome(ARDS).

A method wherein the lung involves pulmonary edema.

A method wherein the pulmonary edema is reduced after treatment.

A method wherein the lung injury involves increased pulmonarypermeability.

A method wherein the pulmonary permeability is reduced after treatment.

A method wherein the lung injury involves reduced pulmonary barrierfunction.

A method wherein the reduced pulmonary barrier function is improvedafter treatment.

A method wherein the lung injury involves reduced pulmonary barrier cellsurvival.

A method wherein the reduced pulmonary barrier cell survival is improvedafter treatment.

A method wherein the time the patient is on a ventilator is reducedafter treatment.

A method wherein blood oxygen levels of the patient are returned towithin a normal range after treatment.

A method wherein the patient exhibits an increase in rheumatoid factor(RF) production from neutrophils or an increase in reactive oxygenspecies (ROS) after treatment.

A method wherein the time to recover for the patient is decreased aftertreatment.

A method wherein the viral infection is caused by the Severe AcuteRespiratory Syndrome Corona Virus 2 (SARS-CoV-2).

A method wherein the composition is administered from the groupconsisting of about four times per day, about three times per day, abouttwo times per day, about one time per day, about one time every otherday, about two times per week, and about one time per week.

A method wherein the composition is administered about one time per day.

A method wherein the composition is administered about two times perday.

A method wherein the composition is administered about three times perday.

A method wherein the composition is administered about four times perday.

A method wherein the composition is administered until the infection istreated.

A method wherein the composition is administered for a period from thegroup consisting of about 1 day to about 30 days [or about one month],about 1 day to about 14 days, about 1 day to about 10 days, about 1 dayto about 1 week (7 days), about 1 day to about 5 days, or about 1 day toabout 3 days.

A method wherein the composition is administered for a period from about1 day to about 14 days.

A method wherein at least one of the following pharmacokineticparameters achieved in the patient is selected from an AUC of about1,037 mcg*h/mL or a Cmax of about 58.1 mcg/mL (equivalent to about 132μM) for the compound capable of inhibiting MAP3K2/MAP3K3.

A method wherein the composition comprises from about 1 to about 1000 mgper unit dosage of the compound capable of inhibiting MAP3K2/MAP3K3based on the active moiety of the compound.

A method wherein the composition comprises about 100 mg, or about 200mg, or about 300 mg, or about 400 mg, or about 500 mg or about 600 mg,or about 700 mg, or about 800 mg per unit dosage of the compound capableof inhibiting MAP3K2/MAP3K3 based on the active moiety of the compound.

A method further comprising an additional active agent.

A method wherein the additional agent is remdesivir, or apharmaceutically acceptable salt, ester, solvate, or prodrug thereof.

A pharmaceutical composition for treating, preventing, or reducing theseverity of a lung injury associated with a coronavirus infection,comprising:

(a) a therapeutically effective amount of a compound capable ofinhibiting MAP3K2/MAP3K3, or a pharmaceutically acceptable salt, ester,solvate, or prodrug thereof, and(b) a pharmaceutically acceptable carrier.

A composition wherein the compound capable of inhibiting MAP3K2/MAP3K3is selected from the group consisting of pazopanib or nintendanib, andcombinations thereof, or a pharmaceutically acceptable salt, solvate, orprodrug thereof.

A composition that is an aqueous composition.

A composition that is in the form of a dry powder.

A composition for administration via a route selected from the groupconsisting of nasal, inhalation (i.e. respiratory), intratracheal,intrapulmonary, and intrabronchial.

A composition further comprising an additional active agent.

A composition wherein the additional agent is remdesivir, or apharmaceutically acceptable salt, ester, solvate, or prodrug thereof.

A device or kit for administration of a composition of the presentinvention.

The use of a compound capable of inhibiting MAP3K2/MAP3K3, or apharmaceutically acceptable salt, ester, solvate, or prodrug thereof, inthe manufacture of a medicament for delivery of a therapeuticallyeffective amount of a pharmaceutical composition comprising a compoundcapable of inhibiting MAP3K2/MAP3K3 for treating, preventing, orreducing the severity of a lung injury associated with a viralinfection.

A use wherein the viral infection is caused by the Severe AcuteRespiratory Syndrome Corona Virus 2 (SARS-CoV-2).

A use wherein said compound capable of MAP3K2/MAP3K3 inhibition isselected from the group consisting of pazopanib or nintedanib, andcombinations thereof, or a pharmaceutically acceptable salt, solvate,ester, or prodrug thereof.

A compound capable of inhibiting MAP3K2/MAP3K3, or a pharmaceuticallyacceptable salt, ester, solvate, or prodrug thereof, for use intreating, preventing, or reducing the severity of a lung injuryassociated with a viral infection when administered to a subject in needthereof in a therapeutically effective amount.

A compound wherein the viral infection is caused by the Severe AcuteRespiratory Syndrome Corona Virus 2 (SARS-CoV-2).

A compound capable of MAP3K2/MAP3K3 selected from the group consistingof pazopanib or nintedanib, and combinations thereof, or apharmaceutically acceptable salt, solvate, ester, or prodrug thereof.

These and other aspects of the present invention will become apparentfrom the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows pulmonary permeability in a mouse coronavirus induced acutelung injury model with three doses of a MAP3K2/MAP3K3 inhibitor aftervirus inoculation;

FIG. 2 shows pulmonary permeability in a mouse coronavirus induced acutelung injury model with two doses of a MAP3K2/MAP3K3 inhibitor aftervirus inoculation;

FIG. 3 shows pulmonary permeability in a mouse coronavirus induced acutelung injury model with one dose of a MAP3K2/MAP3K3 inhibitor given 24hours after virus inoculation;

FIG. 4 shows pulmonary permeability in a mouse coronavirus induced acutelung injury model with one dose of a MAP3K2/MAP3K3 inhibitor given 48hours after virus inoculation;

FIG. 5 shows pulmonary permeability in a mouse coronavirus induced acutelung injury model with one dose of a MAP3K2/MAP3K3 inhibitor given 72hours after virus inoculation;

FIG. 6 shows probability of survival in a mouse coronavirus inducedacute lung injury model with daily doses of a MAP3K2/MAP3K3 inhibitor.

FIG. 7 shows body weight loss in a mouse coronavirus induced acute lunginjury model with daily doses of a MAP3K2/MAP3K3 inhibitor.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the following terms and abbreviations have the indicatedmeanings unless expressly stated to the contrary.

The term “compound capable of inhibiting MAP3K2/MAP3K3” means a compoundthat is an inhibitor of MAP3K2 and/or MAP3K3, i.e. a MAP3K2/MAP3k3inhibitor. The inhibition of MAP3K2 and/or MAP3K3 can be complete orpartial.

The terms “effective”, “pharmaceutically effective”, and“therapeutically effective” means an amount of a MAP3K2/MAP3K3 inhibitorneeded to provide a meaningful or demonstrable benefit, as understood bymedical practitioners, to a subject, such as a human patient in need oftreatment. Conditions, intended to be treated include, for example,coronavirus infections, including lung injury. For example, a meaningfulor demonstrable benefit can be assessed or quantified using variousclinical parameters. The demonstration of a benefit can also includethose provided by models, including but not limited to in vitro models,in vivo models, and animal models.

The term “inhalation” as used herein also includes the term“respiratory” and means administration within the respiratory tract byinhaling orally or nasally for local or systemic effect.

The term “intranasal” as used herein with respect to the pharmaceuticalcompositions and actives therein, means a composition that isadministered through the nose for delivery across the mucosal membraneinside the nasal cavity. This membrane is a well vascularized thinmucosa. Furthermore, this mucosa is in close proximity to the brain andprovides a means to maximize the transport of drugs across theblood-brain barrier. The blood-brain barrier is a highly selectivesemipermeable border that separates the circulating blood from the brainand extracellular fluid in the central nervous system. Althoughintranasal administration can be employed for the present invention, itcan be preferable in other circumstances to provide delivery directly tothe lungs by inhalation/respiratory, or other suitable means.

The U.S. Food & Drug Administration has provided a standard for allroutes of administration for drugs, i.e. “Route of Administration”.Examples of selected routes that are especially suitable for the presentinvention are shown below.

NCI* SHORT FDA CONCEPT NAME DEFINITION NAME CODE ID INTRABRONCHIALAdministration within a I-BRONCHI 067 C38225 bronchus. INTRAPERITONEALAdministration within the I-PERITON 004 C38258 peritoneal cavity.INTRAPLEURAL Administration within the I-PLEURAL 043 C38259 pleura.INTRAPULMONARY Administration within the I-PULMON 414 C38261 lungs orits bronchi. INTRAVENOUS Administration within or into IV 002 C38276 avein or veins. LARYNGEAL Administration directly upon LARYN 364 C38282the larynx. NASAL Administration to the nose; NASAL 014 C38284administered by way of the nose. ORAL Administration to or by way ORAL001 C38288 of the mouth. OROPHARYNGEAL Administration directly to ORO410 C38289 the mouth and pharynx. RESPIRATORY Administration within theRESPIR 136 C38216 (INHALATION) respiratory tract by inhaling orally ornasally for local or systemic effect. TOPICAL Administration to a TOPIC011 C38304 particular spot on the outer surface of the body. TRANSDERMALAdministration through the T-DERMAL 358 C38305 dermal layer of the skinto the systemic circulation by diffusion. TRANSMUCOSAL Administrationacross the T-MUCOS 122 C38283 mucosa. *National Cancer Institutewww.fda.gov/drugs/data-standards-manual-monographs/route-administration

The term “pharmaceutically acceptable” is used herein with respect tothe compositions, in other words the formulations, of the presentinvention, and also with respect to the pharmaceutically acceptablesalts, esters, solvates, and prodrugs thereof. The pharmaceuticalcompositions of the present invention comprise a therapeuticallyeffective amount of the active agents and a pharmaceutically acceptablecarrier. These carriers can contain a wide range of excipients.Pharmaceutically acceptable carriers are those conventionally knowncarriers having acceptable safety profiles. The compositions are madeusing common formulation techniques. See, for example, Remington'sPharmaceutical Sciences, 17th edition, edited by Alfonso R. Gennaro,Mack Publishing Company, Easton, Pa., 17th edition, 1985. Regardingpharmaceutically acceptable salts, these are described below.

The term “subject” means a human patient or animal in need of treatmentor intervention for a coronavirus infection.

The terms “treating” and its derivatives such as “treat” or “treatment,”as used herein, may be used with respect to a particular condition, forexample, lung injury due to or associated with a viral infection such asa coronavirus infection, including but not limited to, acute lung injury(ALI), lung fibrosis, and/or acute respiratory distress syndrome (ARDS).In reference to a particular condition, “treating” and its derivativesare inclusive of several meanings, including (1) to alleviate one ormore symptoms, effects, or side effects associated with the condition,(2) to ameliorate the condition and/or one or more of the biologicalmanifestations or underlying causes of the condition, (3) to interferewith one or more of the biological manifestations or underlying causesof the condition or with one or more points in the biological cascade(s)associated with the condition, (4) to slow the progression of, or arrestthe development of, the condition or of one or more of the biologicalmanifestations of the condition, (5) to prevent or reduce the risk of asubject developing the condition, in some cases prophylactically whenthe subject has one or more risk factors for the condition or has beenexposed to or infected with a virus being associated or having potentialto cause the condition (6) to cause regression of the condition, orimprovement or reversal of, the biological manifestations or underlyingcauses of the conditions. It can be appreciated that “treating” mayencompass one or more of these meanings simultaneously and that asubject's condition may change over time or throughout the course oftreatment such that the meaning of “treating” as applied to a givensubject may change over time or throughout the course of treatment.“Treatment” could be in combination with other therapies or alone.

Methods

The methods of treatment using a MAP3K2/MAP3K3 inhibitor or apharmaceutically acceptable salt, solvate ester, or prodrug thereof orthe pharmaceutical compositions of the present invention, in variousembodiments also include the use of a MAP3K2/MAP3K3 inhibitor or apharmaceutically acceptable salt, solvate ester, or prodrug thereof inthe manufacture of a medicament for the desired treatment, such as fortreating or preventing lung injury due to or associated with a COVID-19infection. Also provided for under methods of treatment are compoundscapable of inhibiting MAP3K2/MAP3K3, or a pharmaceutically acceptablesalts, esters, solvates, or prodrugs thereof, for use in treating,preventing, or reducing the severity of a lung injury due to orassociated with a viral infection when administered to a subject in needthereof in a therapeutically effective amount.

The lung injury can be a manifestation of an inflammatory response andcan be characterized by severe hypoxemia and a substantial reduction inpulmonary compliance due to diffuse alveolar damage, neutrophilicinflammation, and protein-rich edema in the lungs. A consequence of thisdamage is edema and reduced blood oxygen levels, as the affected lungsare less able to effectively transfer oxygen to the bloodstream and toremove carbon dioxide from it.

Surprisingly, it has been found that the compounds of the presentinvention increase rheumatoid factor (RF) production from neutrophils.Even though the neutrophils themselves are harmful, the RF factorincrease is a good thing because it helps the tissue to recover.Neutrophils which are the most abundant leukocytes in the circulatorysystem, are present in acute lung injury. Although they play animportant role in the immune system and the immune response, theycontribute to inflammation-related tissue damage, because of theirnatural functions of phagocytosis, enzyme release, and the release ofreactive oxygen species, which is generally believed to exacerbatetissue damage. However, previously it had been shown that increasedreactive oxygen species (ROS) production from neutrophils by MAP3K2and/or MAP3K3 inhibition can protect the lung during acute injury. SeeWO 2018/195084, to Wu et al., published Oct. 25, 2018, and Yuan, Q. etal. “Pazopanib ameliorates acute lung injuries via inhibition of MAP2K2and MPA3K3”, Science Translational Medicine, 13, eabc2499, publishedApr. 28, 2021.

The inhibition of MAP3K2 and MAP3K3 can lead to an increase in ROSproduction in neutrophils to attenuate lung injury. The protein kinaseinhibitor, pazopanib, a small-molecule anticancer drug can inhibitMAP3K2/3 and elevates ROS levels. A possible mechanism of action is thatthe ROS released can be converted to hydrogen peroxide, which can beprotective in pulmonary vasculature integrity, thereby reducing tissuepermeability and edema from lung injury. There thus seems to be adelicate balance to release enough ROS to activate protective Akt (i.e.protein kinase) phosphorylation but not enough to cause irreversibledamage.

In some embodiments, a lung injury may involve edema or pulmonary edema.In some embodiments, methods of treatment using a MAP3K2/MAP3K3inhibitor may reduce, improve, treat, or ameliorate edema or pulmonaryedema. In some embodiments, a lung injury may involve increasedpulmonary permeability. In some embodiments, methods of treatment usinga MAP3K2/MAP3K3 inhibitor may reduce, improve, treat, or ameliorateedema or pulmonary permeability. In some embodiments, a lung injury mayinvolve reduced pulmonary barrier function and/or reduced pulmonarybarrier cell survival. In some embodiments, methods of treatment using aMAP3K2/MAP3K3 inhibitor may increase, improve, treat, or amelioratepulmonary barrier function and/or pulmonary barrier cell survival.

MAP3K2/MAP3K3 Inhibitors

The present invention utilizes a therapeutically effective amount of acompound capable of inhibiting MAP3K2/MAP3K3, or a pharmaceuticallyacceptable salt, solvate, ester, or prodrug thereof for administrationfor treating or preventing lung injuries associated with a viralinfection, such as those due to, associated with, or caused by acoronavirus infection.

MAP3K2 and MAP3K3 are two highly conserved members of the MEK kinase(MEKK) subgroup of the MAP3K superfamily. These enzymes contain a kinasedomain in the C terminus and a PB1 domain near the N terminus. Thekinase domains of MAP3K2 and MAP3K3 share 94% sequence identity, andthese two kinases are expected to share substrates. Transient expressionof the kinases in vitro leads to their auto-activation and activation ofERK1 and ERK2, p38, JNK, and ERKS. In mice, these kinases are involvedin cardiovascular development, lymphocyte differentiation and NF-kappaBregulation.

MAP3K2—Mitogen-activated protein kinase 2 is an enzyme that in humans isencoded by the MAP3K2 gene. This kinase preferentially activates otherkinases involved in the MAP kinase signaling pathway. This kinase hasbeen shown to directly phosphorylate and activate IkappaB kinases, andthus plays a role in NF-kappa B signaling pathway. This kinase has alsobeen found to bind and activate protein kinase C-related kinase 2, whichsuggests its involvement in a regulated signaling process.

MAP3K3—Mitogen-activated protein kinase 3 is an enzyme that in humans isencoded by the MAP3K3 gene, which is located on the long arm ofchromosome 17 (17q23.3).] This gene product is a 626-amino acidpolypeptide.

Coronaviruses are a group of related RNA viruses that cause diseases inmammals and birds. In humans, these viruses cause respiratory tractinfections that can range from mild to lethal. By late 2020 to early2021, several vaccines were given FDA Emergency Use Authorization andone treatment, remdesivir, is FDA approved. Other known treatmentsinclude convalescent plasma, and biologics such as recombinantantibodies.

Coronaviruses contain a positive-sense single-stranded RNA genome and anucleocapsid of helical symmetry in a protein shell. The virus hasspiked projections which in electron micrographs gives the virus animage analogous to that of the sun and its corona.

It has been found in the present invention that protein kinaseinhibitors having MAP3K2/MAP3K3 inhibition activity can be useful fortreating and preventing coronavirus infections, and more specificallyfor treating or preventing lung injury due to or associated with theinfection, such as acute lung injury (ALI), lung fibrosis, and acuterespiratory distress syndrome (ARDS). Compounds in this class aregenerally useful as anticancer agents. Examples of these compoundsinclude, pazopanib or nintedanib, or a pharmaceutically acceptable salt,solvate, or prodrug thereof.

However, based on the prescribing information and label warnings forcompounds, such as pazopanib, one of ordinary skill in the art might nothave been motivated to select them for the treatment and prevention ofcoronavirus infections, nor for treating or preventing associated lunginjury. This information would seem to teach away from the presentinvention to use pazopanib, and also other active compounds that areprotein kinase inhibitors or that have the potential for MAP3K2/MAP3K3inhibition.

For example, for pazopanib there is the warning that the drug “may causelung problems that may lead to death” and the patient instructions to“Nell your health care provider right away if you develop a cough thatwill not go away or have shortness of breath.” This warning would seemto teach away from the present invention to use pazopanib, or even otheractive compounds that are protein kinase inhibitors or that have thepotential for MAP3K2/MAP3K3 inhibition. Additional warnings include“[s]erious infections” and patient instructions to “[c]all your healthcare provider if you experience fever; cold symptoms, such as runny noseor a sore throat that does not go away; flu symptoms, such as cough,feeling tired, and body aches . . . ” Reported side effects include“trouble breathing”.

Other warnings provided for pazopanib include the following:

“Interstitial Lung Disease (ILD)/Pneumonitis: ILD/pneumonitis, which canbe fatal, has been reported in 0.1% of patients in the clinical trials .. . Monitor patients for ILD/pneumonitis, and discontinue . . . ifsymptoms of ILD or pneumonitis develop.”

“Infection: Serious infections (with or without neutropenia), some withfatal outcomes, have been reported. Monitor for signs and symptoms, andtreat active infection promptly. Consider interruption ordiscontinuation.”

“Pneumothorax [i.e., lung collapse]: [It has been reported that [t]wo of290 patients treated . . . and no patients on the placebo arm in therandomized RCC trial developed a pneumothorax. In the randomized STStrial, pneumothorax occurred in 3% (8/240) of patients treated . . . andin no patients on the placebo arm.

See the HIGHLIGHTS OF PRESCRIBING INFORMATION, VOTRIENT, May 2017; andwww.hcp.novartis.com/products/votrient/?site=VRT-1218078GK100252&source=01030&gclid=EAIalQobChMI4obj5KvD6QIVklbACh00EAS4EAAYASAAEgKnRvD_BwE&gclsrc=aw.ds.

Examples of MAP3K2/MAP3K3 inhibitors useful herein include thoseselected from the group consisting of pazopanib or nintedanib,combinations thereof, or a pharmaceutically acceptable salt, solvate,ester, or prodrug thereof.

See U.S. Pat. No. 8,114,885, to Boloor et al., issued Feb. 14, 2012;U.S. Pat. No. 7,858,626, to Boloor et al., issued Dec. 28, 2010; U.S.Pat. No. 7,262,203, to Boloor et al., issued Aug. 28, 2007; U.S. Pat.No. 7,119,093, to Roth et al., issued Oct. 10, 2006; U.S. Pat. No.7,105,530; to Boloor et al., issued Sep. 12, 2006; U.S. Pat. No.6,762,180, to Roth et al., issued Jul. 13, 2004.

Pazopanib

The present invention utilizes a therapeutically effective amount of theMAP3K2/MAP3K3 inhibitor pazopanib, or a pharmaceutically acceptablesalt, ester, solvate, or prodrug thereof, and also a pharmaceuticallyacceptable carrier, for treating or preventing lung injury due to orassociated with an infection such as a coronavirus infection.

The structure of pazopanib has a pyrimidine moiety. Pazopanib is used asits hydrochloride salt for treatment of kidney cancer. It also has arole as an antineoplastic agent, a tyrosine kinase inhibitor, a vascularendothelial growth factor receptor antagonist and an angiogenesismodulating agent. Pazopanib is a member of indazoles, an aminopyrimidineand a sulfonamide and is a conjugate base of a pazopanib(1+).

Pazopanib corresponds to the following chemical structure:

Pazopanib has the IUPAC name5-({4-[(2,3-Dimethyl-2H-indazol-6-yl)methylamino]pyrimidin-2-yl}amino)-2-methylbenzenesulfonamideand corresponds to the CAS Registry Number 444731-52-6 and ChemSpiderNumber 9700526. Pazopanib has the molecular formula C₂₁H₂₃N₇O₂S and hasa molecular weight (molar mass) of 437.5 g/mol. Pazopanib is usuallydelivered as the hydrochloride salt and has a molecular weight (i.e. amolar mass) for the hydrochloride salt of 1473.977 grams/mole.Therefore, 216.7 mg of pazopanib hydrochloride would be equivalent to200 mg of pazopanib. Note that these molecular weight values will varyslightly depending on what atomic weight values are used for thecalculations. Pazopanib is reported to have a half-life of 30.9-31.9hours, a bioavailability of 14-21%, and protein binding of >99.5%.

Pazopanib was originally identified as a small molecule inhibitor ofmultiple protein tyrosine kinases with potential antineoplasticactivity. Pazopanib selectively inhibits vascular endothelial growthfactor receptors (VEGFR)-1, -2 and -3, c-kit and platelet derived growthfactor receptor (PDGF-R), which may result in inhibition of angiogenesisin tumors in which these receptors are upregulated. It is used in thetherapy of advanced renal cell carcinoma and soft tissue sarcomas.Pazopanib therapy is commonly associated with transient elevations inserum aminotransferase during therapy and has been linked to rare, butoccasionally severe and even fatal cases of clinically apparent acuteliver injury. Pazopanib is known by the trade name Votrient® and ismarketed by Novartis, where it is available as a 200 mg oral tablet.Other code names associated with pazopanib include GW-786034 andUNII-7RN5DR86CK. For cancer therapy, Votrient is administered once dailywithout food (at least 1 hour before or 2 hours after a meal) once dailyat a dose of 800 mg.

Pharmaceutically acceptable salts, esters, solvates, and prodrugs ofpazopanib are useful for the methods and compositions of the presentinvention. As used herein, “pharmaceutically acceptable salts, esters,solvates and prodrugs” refer to derivatives of pazopanib. Examples ofpharmaceutically acceptable salts include, but are not limited to, thehydrochloride salt, the hydrobromide salt, the hydroiodide salt, thehydrogen sulfate salt. As described above, the hydrochloride salt ofpazopanib is currently marketed.

The pharmaceutically acceptable salts, esters, and prodrugs of pazopanibcan be prepared from the parent compound by conventional chemicalmethods. Generally, the salts can be prepared by reacting the free baseform of the compound with a stoichiometric amount of the appropriatestrong acid in water or in an organic solvent, or in a mixture of thetwo; generally, non-aqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred.

The prodrugs of pazopanib can be prepared using convention chemicalmethods, depending on the prodrug chosen. A prodrug is a medication orcompound that, after administration, is metabolized (i.e., convertedwithin the body) into a pharmacologically active drug. Prodrugs can bedesigned to improve bioavailability when a drug itself is poorlyabsorbed from the gastrointestinal tract. Prodrugs are intended toinclude covalently bonded carriers that release an active parent drug ofthe present invention in vivo when such prodrug is administered.

Nintedanib

The present invention utilizes a therapeutically effective amount ofnintedanib, or a pharmaceutically acceptable salt, ester, solvate, orprodrug thereof, and also a pharmaceutically acceptable carrier, fortreating or preventing lung injury due to or associated with a viralinfection, such as a coronavirus infection. Nintedanib is a tyrosinekinase inhibitor, and the present invention provides evidence for itsuse as a MAP3K2/MAP3K3 inhibitor.

The structure of nintedanib has an oxo-indole. Nintedanib is usuallyadministered in oral form as its ethanesulfonic acid salt, i.e. theesylate. Nintedanib is sold under the brand names Ofev and Vargatef, andis marketed by Boehringer Ingelheim Pharmaceuticals, Inc. Nintedanib isprescribed for the treatment of idiopathic pulmonary fibrosis and alongwith other medications for some types of non-small-cell lung cancer.

Nintedanib corresponds to the following chemical structure:

Nintedanib has the IUPAC name Methyl(3Z)-3-{[(4-{methyl[(4-methylpiperazin-1-yl)acetyl]amino}phenyl)amino](phenyl)methylidene}-2-oxo-2,3-dihydro-1H-indole-6-carboxylateand corresponds to the CAS Registry Number 656247-17-5 and ChemSpiderNumber 7985471. Nintedanib has the molecular formula C₃₁H₃₃N₅O₄ and hasa molecular weight (molar mass) of 539.6248 g/mol. Note that themolecular weight value will vary slightly depending on what atomicweight values are used for the calculations. Nintedanib is reported tohave an elimination half-life of 10-15 hours, a bioavailability of 4.7%,and protein binding of 97.8%.

Pharmaceutically acceptable salts, solvates, and prodrugs of nintedanibare useful for the methods and compositions of the present invention. Asused herein, “pharmaceutically acceptable salts, solvates and prodrugs”refer to derivatives of nintedanib. Examples of pharmaceuticallyacceptable salts include, but are not limited to, the hydrochloridesalt, the hydrobromide salt, the hydroiodide salt, the hydrogen sulfatesalt. As described above, the ethane sulfonic acid salt of nintedanib iscurrently marketed.

The pharmaceutically acceptable salts and prodrugs of nintedanib can beprepared from the parent compound by conventional chemical methods.Generally, the salts can be prepared by reacting the free base form ofthe compound with a stoichiometric amount of the appropriate acid inwater or in an organic solvent, or in a mixture of the two; generally,non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, oracetonitrile are preferred.

The prodrugs of nintedanib can be prepared using convention chemicalmethods, depending on the prodrug chosen. A prodrug is a medication orcompound that, after administration, is metabolized (i.e., convertedwithin the body) into a pharmacologically active drug. Prodrugs can bedesigned to improve bioavailability when a drug itself is poorlyabsorbed from the gastrointestinal tract. Prodrugs are intended toinclude covalently bonded carriers that release an active parent drug ofthe present invention in vivo when such prodrug is administered.

Dosages and Treatment Regimens

In some embodiments, the compositions comprises from about 1 to about1000 mg, or from about 10 to about 1000 mg, or from about 50 to about1000 mg, or from about 100 to about 1000 mg, or from about 100 to about900 mg, or from about 100 to about 800 mg, or from about 200 to about800 mg, or from about 200 to about 700 mg, or from about 200 to about600 mg, or from about 200 to about 500 mg, or from about 200 to about400 mg, or from about 200 to about 300 mg per unit dosage of thecompound capable of inhibiting MAP3K2/MAP3K3 based on the active moietyof the compound. In some embodiments, the compositions can compriseabout 1 mg, or about 5 mg, or about 10 mg, or about 25 mg or about 50mg, or about 100 mg, or about 150 mg, or about 200 mg, or about 250 mgor about 300 mg, or about 350 mg or about 400 mg, or about 450 mg, orabout 500 mg, or about 550 mg or about 600 mg, or about 650 mg, or about700 mg, or about 800 mg, or about 900 mg, or about 1000 mg per unitdosage of the compound capable of inhibiting MAP3K2/MAP3K3 based on theactive moiety of the compound.

In some embodiments, a dosing range for the compound capable ofinhibiting MAP3K2/MAP3K3 based on the active moiety of the compound isfrom about 0.01 mg/kg to about 1000 mg/kg of body weight/per day of thesubject.

The dosage can be varied to achieve an amount of the active ingredientthat is effective for obtaining the desired therapeutic effect.

The target indication of the invention composition is related to methodsof treating or preventing lung injury due to or associated withcoronavirus infections. The compositions of the present invention can beadministered according to a variety of regimens.

For example, the composition is administered from the group consistingof about four times per day, about three times per day, about two timesper day, about one time per day, about one time every other day, abouttwo times per week, and about one time per week. Furthermore, thecomposition can be administered about one time per day, or about twotimes per day, or about three times per day, or about four times perday.

The composition is administered until the infection is treated. Periodsof administration include those selected from the group consisting ofabout 1 day to about 30 days [or about one month], about 1 day to about14 days, about 1 day to about 10 days, about 1 day to about 1 week (7days), about 1 day to about 5 days, or about 1 day to about 3 days.

It is also desirable to achieve appropriate pharmacokinetic and/orpharmacodynamic properties. The methods and compositions in someembodiments demonstrate at least one of the following pharmacokineticparameters in the patient: an AUC of about 1,037 mcg*h/mL or a Cmax ofabout 58.1 mcg/mL (equivalent to about 132 μM) for the compound capableof inhibiting MAP3K2/MAP3K3.

Combination Therapies

In other embodiments, the patient or subject can be administered atleast one additional active agent for treating, preventing or reducingthe severity of the lung injury, or to treat, prevent, or reduce theseverity of the viral infection or other disease manifestations orsymptoms.

In some embodiment, the compositions and methods of the presentinvention can be provided in combination with the anti-viral agentremdesivir. Remdesivir is an investigational broad-spectrum antiviralmedication developed by Gilead Sciences, Inc. that is currently beingevaluated as a treatment for COVID-19. It has been authorized foremergency use in the United States and approved for use in Japan. It hasthe IUPAC chemical name(2S)-2-{(2R,3S,4R,5R)[5-(4-Aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxy-tetrahydro-furan-2-ylmethoxy]phenoxy-(S)-phosphorylamino}propionic acid 2-ethyl-butyl ester, CAS Registry Number1809249-37-3 and ChemSpider Number 58827832. Remdesivir corresponds tothe chemical formula C₂₇H₃₅N₆O₈P, and has a molar mass of 602.585 g/mol.

Formulations

In the present invention other optional ingredients may also beincorporated into the nasal delivery system provided they do notinterfere with the action of the drug or significantly decrease theabsorption of the drug across the nasal mucosa. Such ingredients caninclude, for example, pharmaceutically acceptable excipients andpreservatives. The excipients that can be used in accordance with thepresent invention include, for example, bio-adhesives and/orswelling/thickening agents.

In the present invention, any other suitable absorption enhancers asknown in the art may also be used.

Preservatives can also be added to the present compositions. Suitablepreservatives that can be used with the present compositions include,for example, benzyl alcohol, parabens, thimerosal, chlorobutanol andbenzalkonium, with benzalkonium chloride being preferred. Typically, thepreservative will be present in the present compositions in aconcentration of up to about 2% by weight. The exact concentration ofthe preservative, however, will vary depending upon the intended use andcan be easily ascertained by one skilled in the art.

The absorption enhancing agent includes (i) a surfactant; (ii) a bilesalt (including sodium taurocholate); (iii) a phospholipid additive,mixed micelle, or liposome; (iv) an alcohol (including a polyol asdiscussed above, for example, propylene glycol or polyethylene glycolsuch as PEG 3000, etc.); (v) an enamine; (vi) a nitric oxide donorcompound; (vii) a long-chain amphipathic molecule; (viii) a smallhydrophobic uptake enhancer; (ix) sodium or a salicylic acid derivative;(x) a glycerol ester of acetoacetic acid; (xi) a cyclodextrin orcyclodextrin derivative; (xii) a medium-chain or short-chain (e.g. Cl toC 12) fatty acid; and (xiii) a chelating agent; (xiv) an amino acid orsalt thereof; and (xv) an N-acetylamino acid or salt thereof.

Solubility enhancers may increase the concentration of the drug orpharmaceutically acceptable salt thereof in the formulation. Usefulsolubility enhancers include, e.g., alcohols and polyalcohols.

An isotonizing agent may improve the tolerance of the formulation in anasal cavity. A common isotonizing agent is NaCl. Preferably, when theformulation is an isotonic intranasal dosage formulation, it includesabout 0.9% NaCl (v/v) in the aqueous portion of the liquid carrier.

The thickeners may improve the overall viscosity of the composition,preferably to values close to those of the nasal mucosa. Suitablethickeners include methylcellulose, carboxymethylcellulose,polyvinylpyrrolidone, sodium alginate, hydroxypropylmethylcellulose, andchitosan.

A humectant or anti-irritant improves the tolerability of thecomposition in repeated applications. Suitable compounds include, e.g.glycerol, tocopherol, mineral oils, and chitosan.

Various additional ingredients can be used in the compositions of thepresent invention. The compositions can comprise one or more furtheringredients selected from a preservative, an antioxidant, an emulsifier,a surfactant or wetting agent, an emollient, a film-forming agent, or aviscosity modifying agent. These components can be employed and used atlevels appropriate for the formulation based on the knowledge of onewith ordinary skill in the pharmaceutical and formulation arts. Theamounts could range from under 1 percent by weight to up to 90 percentor even over 99 percent by weight.

In another aspect, suitable propellants can be used for dispensing theproduct when in the form of a liquid or powder for delivery from a spraydevice or nebulizer.

In one aspect, a preservative can be included. In another aspect, anantioxidant can be included. In another aspect, an emulsifier can beincluded. In another aspect, an emollient can be included. In anotheraspect, a viscosity modifying agent can be included. In another aspect,a surfactant or wetting agent can be included. In another aspect, a filmforming agent can be included. In another aspect, the pharmaceuticalcomposition is in the form selected from the group consisting of a gel,ointment, lotion, emulsion, cream, liquid, spray, suspension, jelly,foam, mousse, paste, tape, dispersion, aerosol. These components can beemployed and used at levels appropriate for the formulation based on theknowledge of one with ordinary skill in the pharmaceutical andformulation arts.

In another aspect, the at least one preservative can be selected fromthe group consisting of parabens (including butylparabens,ethylparabens, methylparabens, and propylparabens), acetone sodiumbisulfite, alcohol, benzalkonium chloride, benzethonium chloride,benzoic acid, benzyl alcohol, boric acid, bronopol, butylatedhydroxyanisole, butylene glycol, calcium acetate, calcium chloride,calcium lactate, cetrimide, cetylpyridinium chloride, chlorhexidine,chlorobutanol, chlorocresol, chloroxylenol, cresol, edetic acid,glycerin, hexetidine, imidurea, isopropyl alcohol, monothioglycerol,pentetic acid, phenol, phenoxyethanol, phenylethyl alcohol,phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate,potassium benzoate, potassium metabisulfite, potassium nitrate,potassium sorbate, propionic acid, propyl gallate, propylene glycol,propylparaben sodium, sodium acetate, sodium benzoate, sodium borate,sodium lactate, sodium metabisulfite, sodium propionate, sodium sulfite,sorbic acid, sulfur dioxide, thimerosal, zinc oxide, andN-acetylcysteine, or a combination thereof. These components can beemployed and used at levels appropriate for the formulation based on theknowledge of one with ordinary skill in the pharmaceutical andformulation arts. The amounts could range from under 1 percent by weightto up to 30 percent by weight.

In another aspect, the at least one antioxidant can be selected from thegroup consisting of acetone sodium bisulfite, alpha tocopherol, ascorbicadd, ascorbyl palmitate, butylated hydroxyanisole, butylatedhydroxytoluene, citric acid monohydrate, dodecyl gallate, erythorbicacid, fumaric acid, malic acid, mannitol, sorbitol, monothioglycerol,octyl gallate, potassium metabisulfite, propionic acid, propyl gallate,sodium ascorbate, sodium formaldehyde sulfoxylate, sodium metabisulfite,sodium sulfite, sodium thiosulfate, sulfur dioxide, thymol, vitamin Epolyethylene glycol succinate, and N-acetylcysteine, or a combinationthereof. These components can be employed and used at levels appropriatefor the formulation based on the knowledge of one with ordinary skill inthe pharmaceutical and formulation arts. The amounts could range fromunder 1 percent by weight to up to 30 percent by weight.

In another aspect, the at least one emulsifier can be selected from thegroup consisting of acacia, agar, ammonium alginate, calcium alginate,carbomer, carboxymethylcellulose sodium, cetostearyl alcohol, cetylalcohol, cholesterol, diethanolamine, glyceryl monooleate, glycerylmonostearate, hectorite, hydroxypropyl cellulose, hydroxypropyl starch,hypromellose, lanolin, lanolin alcohols, lauric acid, lecithin, linoleicacid, magnesium oxide, medium-chain triglycerides, methylcellulose,mineral oil, monoethanolamine, myristic acid, octyldodecanol, oleicacid, oleyl alcohol, palm oil, palmitic acid, pectin, phospholipids,poloxamer, polycarbophil, polyoxyethylene alkyl ethers, polyoxyethylenecastor oil derivatives, polyoxyethylene sorbitan fatty acid esters,polyoxyethylene stearates, polyoxyl 15 hydroxystearate,polyoxyglycerides, potassium alginate, propylene glycol alginate,propylene glycol dilaurate, propylene glycol monolaurate, saponite,sodium borate, sodium citrate dehydrate, sodium lactate, sodium laurylsulfate, sodium stearate, sorbitan esters, starch, stearic acid, sucrosestearate, tragacanth, triethanolamine, tromethamine, vitamin Epolyethylene glycol succinate, wax, and xanthan gum, or a combinationthereof. These components can be employed and used at levels appropriatefor the formulation based on the knowledge of one with ordinary skill inthe pharmaceutical and formulation arts. The amounts could range fromunder 1 percent by weight to up to 30 percent by weight.

In another aspect, the at least one emollient can be selected from thegroup consisting of almond oil, aluminum monostearate, butyl stearate,canola oil, castor oil, cetostearyl alcohol, cetyl alcohol, cetylpalmitate, cholesterol, coconut oil, cyclomethicone, decyl oleate,diethyl sebacate, dimethicone, ethylene glycol stearates, glycerin,glyceryl monooleate, glyceryl monostearate, isopropyl isostearate,isopropyl myristate, isopropyl palmitate, lanolin, lanolin alcohols,lecithin, mineral oil, myristyl alcohol, octyldodecanol, oleyl alcohol,palm kernel oil, palm oil, petrolatum, polyoxyethylene sorbitan fattyacid esters, propylene glycol dilaurate, propylene glycol monolaurate,safflower oil, squalene, sunflower oil, tricaprylin, triolein, wax,xylitol, zinc acetate, or a combination thereof. These components can beemployed and used at levels appropriate for the formulation based on theknowledge of one with ordinary skill in the pharmaceutical andformulation arts. The amounts could range from under 1 percent by weightto up to 60 percent by weight.

In another aspect, the at least one viscosity modifying agent can beselected from the group consisting of acacia, agar, alginic acid,aluminum monostearate, ammonium alginate, attapulgite, bentonite,calcium alginate, calcium lactate, carbomer, carboxymethylcellulosecalcium, carboxymethylcellulose sodium, carrageenan, cellulose,ceratonia, ceresin, cetostearyl alcohol, cetyl palmitate, chitosan,colloidal silicon dioxide, corn syrup solids, cyclomethicone,ethylcellulose, gelatin, glyceryl behenate, guar gum, hectorite,hydrophobic colloidal silica, hydroxyethyl cellulose, hydroxyethylmethylcellulose, hydroxypropyl cellulose, hydroxypropyl starch, hypromellose,magnesium aluminum silicate, maltodextrin, methylcellulose, myristylalcohol, octyldodecanol, palm oil, pectin, polycarbophil, polydextrose,polyethylene oxide, polyoxyethylene alkyl ethers, polyvinyl alcohol,potassium alginate, propylene glycol alginate, pullulan, saponite,sodium alginate, starch, sucrose, sugar, sulfobutylether β-cyclodextrin,tragacanth, trehalose, and xanthan gum, or a combination thereof. Thesecomponents can be employed and used at levels appropriate for theformulation based on the knowledge of one with ordinary skill in thepharmaceutical and formulation arts. The amounts could range from under1 percent by weight to up to 60 percent.

In another aspect, the at least one film forming agent can be selectedfrom the group consisting of ammonium alginate, chitosan, colophony,copovidone, ethylene glycol and vinyl alcohol grafted copolymer,gelatin, hydroxypropyl cellulose, hypromellose, hypromellose acetatesuccinate, polymethacrylates, poly(methyl vinyl ether/maleic anhydride),polyvinyl acetate dispersion, polyvinyl acetate phthalate, polyvinylalcohol, povidone, pullulan, pyroxylin, and shellac, or a combinationthereof. These components can be employed and used at levels appropriatefor the formulation based on the knowledge of one with ordinary skill inthe pharmaceutical and formulation arts. The amounts could range fromunder 1 percent by weight to up to 90 percent or even over 99 percent byweight.

In another aspect, the at least one surfactant or wetting agent can beselected from the group consisting of docusate sodium, phospholipids,sodium lauryl sulfate, benzalkonium chloride, cetrimide, cetylpyridiniumchloride, alpha tocopherol, glyceryl monooleate, myristyl alcohol,poloxamer, polyoxyethylene alkyl ethers, polyoxyethylene castor oilderivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylenestearates, polyoxyl 15 hydroxystearate, polyoxyglycerides, propyleneglycol dilaurate, propylene glycol monolaurate, sorbitan esters, sucrosestearate, tricaprylin, and vitamin E polyethylene glycol succinate, or acombination thereof. These components can be employed and used at levelsappropriate for the formulation based on the knowledge of one withordinary skill in the pharmaceutical and formulation arts. The amountscould range from under 1 percent by weight to up to 30 percent byweight.

In another aspect, a buffering agent can be included. In another aspect,an emollient can be included. In another aspect, an emulsifying agentcan be included. In another aspect, an emulsion stabilizing agent can beincluded. In another aspect, a gelling agent can be included. In anotheraspect, a humectant can be included. In another aspect, an ointment baseor oleaginous vehicle can be included. In another aspect, a suspendingagent can be included. In another aspect an acidulant can be included.In another aspect, an alkalizing agent can be included. In anotheraspect, a bioadhesive material can be included. In another aspect, acolorant can be included. In another aspect, a microencapsulating agentcan be included. In another aspect, a stiffening agent can be included.These components can be employed and used at levels appropriate for theformulation based on the knowledge of one with ordinary skill in thepharmaceutical and formulation arts. The amounts could range from under1 percent by weight to up to 90 percent or even over 99 by weight.

One of ordinary skill in the pharmaceutical and formulation arts candetermine the appropriate levels of the essential and optionalcomponents of the compositions of the present invention.

The compositions of the present invention can be in a variety of formsincluding oral and intravenous forms, and also parenteral forms andcompositions for injection. However, because delivery to the lungs ishighly desirable the following routes of administration are preferred:nasal, inhalation (i.e. respiratory), intratracheal, intrapulmonary, andintrabronchial.

The compositions can be in the form of liquids, suspensions or drypowders. These compositions can be delivered into the lungs via anebulizer or atomizer. The present invention also contemplates devicesfor spraying the compositions and kits comprising such a delivery deviceand instructions for use.

Methods of preparing the compositions are also intended as part of thepresent invention and would be apparent to one of ordinary skill in thepharmaceutical and formulation arts using standard formulation andmixing techniques.

Methods of Treatment

The present invention utilizes a therapeutically effective amount of acompound having MAP3K2/MAP3K3 inhibition activity, or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier fortreating or preventing lung injury due to or associated with aninfection such as a coronavirus infection.

The methods may comprise administration to or into the lung of a patientin need thereof. The methods may also comprise any other administrationroute that provides a therapeutically effective and sufficientconcentration of the compound in the lungs and/or systemically, such asoral, oropharyngeal, parenteral, nasal, respiratory (inhalation),intraperitoneal, intrapleural, intravenous, lanyngeal, oropharyngeal,topical, transdermal, transmucosal intratracheal, intrapulmonary, andintrabronchial.

Various dosing regimens can be prescribed and used based on the skilland knowledge of the physician or other practitioner. In someembodiments, a unit dosage of the composition, as described herein canbe applied at least once daily. In other embodiments, a unit dosage ofthe composition can be applied at least twice daily, or at least onceweekly, or at least twice weekly. Based on the pharmacokinetic andpharmacodynamic parameters of the active ingredient, the dosing amountand regimen can be appropriately varied.

Therapy can be continued in the judgment of the physician orpractitioner until the desired therapeutic benefit is achieved. In someinstances, it can be desirable to continue long term or chronic therapy.

The therapy of the present invention is especially important for thosepatients that are hospitalized and critically ill, particularly those inintensive care units. The invention is useful for those patients onventilators and respirators.

In lung injury, a number of factors can be involved. The methods of thepresent invention provide a means for reducing edema, and in maintainand increasing blood oxygen levels in those patients that have beencompromised due to the lung injury. The present methods also provide ameans to increase RF (rheumatoid factor) production from neutrophils.These factors help damaged tissue to recover. Neutrophils withinsynovial fluid are activated by soluble immune complexes (RF, ACPA) thatinduce neutrophil degranulation and release of reactive oxygen species(ROS) and proteases, which lead to degradation of hyaluronic acid andactivation of cytokines.

The compositions can be administered by those routes to most effectivelyhave it get into the lungs. The compositions can be administered viaaerosol generating devices such as nebulizers. The compositions can bepart of a kit for therapeutic treatment. The kits can comprise anapplicator device and also instructional material for using it.

Kits for Providing Compositions and Treatment

The present invention also includes kits for providing the compositionsand methods of treatment. These kits comprise the pharmaceuticalcomposition, instruction materials and prescribing information, and forcertain modes of administration a spray device or nebulizer. In furtherembodiments, the kits can comprise additional active ingredients fortreating, preventing or reducing the severity of the lung injury or formanaging other symptoms or aspects of the viral infection. The kits canalso provide personal protective equipment such as face masks,disposable gloves, and disposable protective garments for healthcareprofessionals and other workers involved with administering thetreatment to the patient.

EXAMPLES

The following examples further describe and demonstrate embodimentswithin the scope of the present invention. The Examples are given solelyfor purpose of illustration and are not to be construed as limitationsof the present invention, as many variations thereof are possiblewithout departing from the spirit and scope of the invention.

Example 1: Investigation of Pazopanib on Lung Injuries

We investigated the effects of the FDA-approved anti-cancer drugpazopanib on lung injuries in a mouse coronavirus-induced pneumonitismode

We have strong data to show that pazopanib is a substrate specificinhibitor of MAP3K2 and 3 and that inhibition of these two proteinkinases ameliorates lung injury, which is believed to be a majorcontributor of death associated with COVID-19. To provide furtherconfidence and justification for carrying out human study of the drug onCOVID-19 patients, we propose to investigate the effects of pazopanib ina mouse model infected with coronavirus MHV-1, which exhibit lung injuryphenotypes similarly to those caused by SARS-CoV.

The current COVID-19 pandemic is caused by a novel coronavirus,designated as severe acute respiratory syndrome coronavirus 2(SARS-CoV-2). The disease causes death of about 2% cases probably due tomassive alveolar damage and progressive respiratory failure. Publishedpulmonary pathology and radiological reports of COVID-19 and previousSARS-CoV or Middle Eastern respiratory syndrome (MERS) provide clearassociation of the disease with acute respiratory distress syndrome(ARDS) [1-4].

ARDS is a severe form of acute lung injury (ALI), which is caused bydirect or indirect insults to the lung [5, 6]. In case of COVID-19, lunginjury is likely caused by both direct and indirect means. SARS-CoV hasbeen shown to cause necrosis of lung epithelial cells (direct insult).In addition, strong inflammatory responses elicited by the viralinfection should also cause lung injury (indirect insult). In additionto viral infection, there are many other causes for ALI/ARDS, includingbacterial infection, gastric acid aspiration, and trauma. The incidenceof ALI/ARDS is reported to be around 200,000 per year in the US(excluding COVID-19) with a mortality rate of around 40% [5]. Currentlythere are no pharmacological interventions for the diseases. Care ofthese conditions is largely dependent on supportive measures [7, 8].This probably contributes to the high fatality rate of COVID-19 and highdemand of respirators.

In our study of the functional roles of two protein kinases MAP3K2 and3, we inadvertently discovered that inhibition of these two kinasesabates acute lung injuries in mouse models involving an unexpectedbeneficial effect of paracrine ROS from myeloid cells (see attachedmanuscript). A hallmark of ALI/ARDS is the abundant presence ofneutrophils in the lungs [9]. This is also true in SARS-CoV-induced ARDS[1, 4]. We found that genetic inactivation of these two kinase homologsincreased ROS production in neutrophils. In addition, the lack of theseMAP3K kinases in myeloid cells attenuated lung injury manifestations andreduced mortality in both direct and indirect lung insult mouse ALImodels. Mechanistically, the kinases phosphorylate the Nox2 subunitp47^(phox) at Ser208 to inhibit p47^(phox) interaction with p22^(phox)and hence Nox2 activity. The importance of Ser208 phosphorylation wasfurther confirmed by its knock-in mutation; hematopoietic loss ofp47^(phox) Ser208 phosphorylation recapitulated the effects of MAP3K2/3loss on neutrophils and ALI. Moreover, we found that inhibition of theMAP3K-p47^(phox) pathway in myeloid cells acted on both pulmonaryvasculature and epithelial cells to enhance the pulmonary barrierfunction and protect lungs from injuries. We subsequently identifiedpazopanib, which is an FDA-approved drug for cancer treatment, as beinga high-affinity (low nanomolar), p47^(phox) substrate-specific inhibitorfor MAP3K2 and 3. Pazopanib was effective in ameliorating ALI in the twomouse ALI/ARDS models in a manner dependent on these two kinases and ROSproduction. Given the drug has been well tolerated in clinic, pazopanibis very likely to be quickly repurposed for treating ALI/ARDS caused bySARS-Cov-2 in this COVID-19 pandemic. The test of pazopanib in a mousecoronavirus infection model that closely resembles human SARS-CoVpulmonary pathology would provide further confidence and justificationfor human study of this drug on COVID-19 patients.

Research Strategy

-   1. Establishment and verification of coronavirus induced SARS-like    pulmonary disease model in mice.

De Albuquerque, et al. reported in 2006 that intranasal infection of A/Jmice with the coronavirus murine hepatitis virus strain 1 (MHV-1)produced ALI/ARDS features of SARS [10]. All MHV-1-infected A/J micedeveloped progressive interstitial edema, neutrophil/macrophageinfiltrates, and hyaline membranes, leading to death of all mice. Weplan to use this model to test prophylactic and therapeutic effects ofpazopanib. The A/J mice will be purchased from Charles River and MHV-1will be purchased from ATCC (ATCC VR-261, the same sources used by DeAlbuquerque, et al.; Jim Macy's lab also has this strain). Viruses willbe first plaque purified and then expanded in murine 17CL1 cells or NCTCclone 1469 (ATCC CCL-9.1). Supernatants are to be collected andsubsequently stored at −80° C. until use. Viral titers are determinedvia plaque assay using the L2 cells (ATCC CCL-149) [10]. We will startwith viral titration test in mice based on what was described by DeAlbuquerque, et al. In that report, mice were infected with 5,000 PFUintranasally, which resulted in ALI phenotypes in two days and death in7-8 days. We will repeat the experiments with 1000, 2500, 5000, 10000PFU. Mice survival will be monitored up to 21 days and H&E (hematoxylinand eosin) staining will be performed on lungs and livers collected atday 0, day 2 and day 6 post-MHV infection. Meanwhile, virus titers inlung, brain, liver and spleen and cytokines (IL-6, IL-10, INF-g, TNF-aand MCP-1) levels in blood and BAL (bronchoalveolar lavage) will bedetermined at day 0, day 2, day 4 and day 6.

Anticipated Outcomes

We expect to see A/J mice develop pulmonary pathological features ofSARS including dense macrophage/neutrophil infiltrates, pulmonary edemaand hyaline membranes. Cytokines levels are expected to be elevated inthe lungs and blood.

-   2. Test the effects of pazopanib on lung injuries in coronavirus    induced SARS-like pulmonary disease model.

Once the proper viral dose is determined, the effects of pazopanib willbe tested in this mouse model. Based on our preliminary data fortreating lung injuries induced by HCl in mice, 3 mg/kgbw for pazopanibintravenous (IV)-injection and 65 mg/kgbw for gavage delivery are bestdoses, so we will start from here and try different doses. Mice will befirst intranasally injected with MHV-1 virus, pazopanib will bedelivered to mice via IV at 3 mg/kgbw or via gavage at 65 mg/kgbw,starting from 12 hr post virus infection followed by every 24 hrdelivery. Control mice will be infected with virus as well and deliveredwith vehicle at the same time points. Mice will be monitored daily forsymptoms of disease, including ruffled fur, tremors and lack ofactivity. Survival rate will be examined and pulmonary permeability willbe determined using FITC-Albumin as what we did in other mouse ALImodel. Histological examination of lungs and livers, viral titers indifferent organs and cytokines measurements will be determined as whatwe described above (section 1). In addition, pharmacokinetics ofpazopanib in blood will be determined using established LC/MS method[11]. Besides therapeutic effect, prophylactic effect of pazopanib willbe evaluated as well. In this case, pazopanib will be delivered 12 hrbefore infection via gavage or 2 hr before infection via IV followed byevery 24 hr delivery and the rest evaluation will be the same as what weplanned above.

Based on the results from the acid-induced direct insult and LPS-inducedindirect insult ALI models we performed, we anticipate that pazopanibtreatment should have effects on pulmonary edema in this MHV-1 infectionmodel. We hope that the drug can impact mortality rate as we have seenwith acid and LPS models. And we also hope to see prophylactic effect ofthis drug on lung injury as what we have observed in our acid and LPSmodels. If any of the results is observed, the drug should bejustifiable for immediate human trial.

REFERENCES

-   1. Tian, S., et al., Pulmonary Pathology of Early-Phase 2019 Novel    Coronavirus (COVID-19) Pneumonia in Two Patients With Lung Cancer. J    Thorac Oncol, 2020.-   2. Ng, D. L., et al., Clinicopathologic, Immunohistochemical, and    Ultrastructural Findings of a Fatal Case of Middle East Respiratory    Syndrome Coronavirus Infection in the United Arab Emirates,    April 2014. Am J Pathol, 2016. 186 (3): p. 652-8.-   3. Ding, Y., et al., The clinical pathology of severe acute    respiratory syndrome (SARS): a report from China. J Pathol, 2003.    200 (3): p. 282-9.-   4. Xu, Z., et al., Pathological findings of COVID-19 associated with    acute respiratory distress syndrome. The Lancet Respiratory    Medicine, 2020.-   5. Johnson, E. R. and M. A. Matthay, Acute Lung Injury:    Epidemiology, Pathogenesis, and Treatment. Journal of Aerosol    Medicine and Pulmonary Drug Delivery, 2010. 23 (4): p. 243-252.-   6. Maca, J., et al., Past and Present ARDS Mortality Rates: A    Systematic Review. Respir Care, 2017. 62 (1): p. 113-122.-   7. Brower, R. G., et al., Ventilation with lower tidal volumes as    compared with traditional tidal volumes for acute lung injury and    the acute respiratory distress syndrome. New England Journal of    Medicine, 2000. 342 (18): p. 1301-1308.-   8. Weinert, C. R., C. R. Gross, and W. A. Marinelli, Impact of    randomized trial results on acute lung injury ventilator therapy in    teaching hospitals. American Journal of Respiratory and Critical    Care Medicine, 2003. 167 (10): p. 1304-1309.-   9. Matthay, M. A. and R. L. Zemans, The acute respiratory distress    syndrome: pathogenesis and treatment. Annu Rev Pathol, 2011. 6: p.    147-63.-   10. De Albuquerque, N., et al., Murine hepatitis virus strain 1    produces a clinically relevant model of severe acute respiratory    syndrome in A/J mice. J Virol, 2006. 80 (21): p. 10382-94.-   11. Minocha, M., V. Khurana, and A. K. Mitra, Determination of    pazopanib (GW-786034) in mouse plasma and brain tissue by liquid    chromatography-tandem mass spectrometry (LC/MS-MS). J Chromatogr B    Analyt Technol Biomed Life Sci, 2012. 901: p. 85-92.

Example 2: Composition for Inhalation Delivery

The following composition is prepared using standard mixing equipmentand procedures.

Ingredient Amount Pazopanib hydrochloride 100 to 800 mg/ml* Water QS toachieve the indicated levels of ingredients *Active amount based onhydrochloride salt

The pazopanib hydrochloride salt is suspended or dissolved in water withgentle mixing.

The composition can be packaged in a spray bottle or nebulizer forinhalation administration.

The composition is useful for treating or preventing lung injury due toor associated with a coronavirus infection.

In another embodiment, the composition of Example 1 is prepared usingnintedanib esylate in place of pazopanib hydrochloride.

Example 3: Efficacy Evaluation of Three Doses of Pazopanib Hydrochloridein Mouse Coronavirus Induced Lung Injury Model Purpose

Evaluate the efficacy of pazopanib hydrochloride composition incoronavirus (Murine Hepatitis Virus Strain 1, MHV-1) induced mouse lunginjury model.

Procedure

a. Pazopanib Composition Preparation

-   -   1. Put 3.294 ml double distilled water (ddH2O) in a small beaker        with a small magnetic stir bar.    -   2. Weigh 1.663 g 2-Hydroxypropyl-beta-cyclodextrin (HP-b-CD).    -   3. Add HP-b-CD slowly into the water while stirring.    -   4. Keep stirring until HP-b-CD completely dissolved.    -   5. Transfer the HP-b-CD solution into a 15 ml tube.    -   6. Weigh 43 mg pazopanib hydrochloride and add it into HP-b-CD        solution.    -   7. Vibrate the tube for 5 min followed by 30 min water bath        sonication.    -   8. Then keep the tube in 50° C. for 30 min and pazopanib        hydrochloride should be completely dissolved.    -   Vehicle Stock: 33.26% (332.6 mg/ml) HP-b-CD in ddH2O    -   Stock solution of pazopanib: 8.6 mg/ml in vehicle control

Vehicle Stock Pazopanib stock Vehicle control   100 ul   0 ul 3 mg/kgbw93.03 ul 6.97 ul * Each mouse is around 20 gbw and retro-orbitalinjection volume is 100 ul

b. Animal procedure

-   -   A/J mice, 8-10 weeks old, were anaesthetized by        Ketamine/Xylazine (100 mg/kg and 10 mg/kg) and were kept under        anesthesia during the whole procedure using Ketamine/Xylazine.        After being deeply anesthetized (assessed by applying a noxious        stimulus, eg toe pinch, and observing no reflex response and no        change in either the rate or character of respiration), mice        received an intranasal inoculation of 5000 PFU MHV-1 in 20 μl        Dulbecco's modified Eagle's medium. After the administration,        the mice were monitored until their breathing gradually returned        to normal. Then the mice were returned to the recovery cage on        the heating pad and monitored for their anesthesia status. Six        hours after the virus inoculation, vehicle control or 3 mg/kgbw        (mg per kg of body weight) pazopanib was delivered to mice via        retro-orbital. Twenty-one hours after the virus inoculation,        vehicle control or 3 mg/kgbw pazopanib was delivered to mice via        retro-orbital again. After another eleven hours, the 3^(rd) dose        of vehicle control or 3 mg/kgbw pazopanib was delivered to mice        via retro-orbital as well. Fifteen hours after the 3rd dose of        the drug administration, 100 μl of FITC-labeled albumin (10        mg/ml) was injected via the retro-orbital vein. Two hours after        FITC-albumin injection, mice were euthanized and bronchoalveolar        lavage (BAL) was collected via instilling 1 ml of        phosphate-buffered saline (PBS) into the lungs, which was        retrieved via a tracheal catheter. The green fluorescent of BAL        was measured by the plate reader. BAL fluorescent intensities        from pazopanib treated mice were normalized to the intensities        from vehicle control-treated mice.

Conclusion

As shown in FIG. 1, treatment with a pharmaceutical composition ofpazopanib can significantly reduce pulmonary permeability in mousecoronavirus induced acute lung injury model when 3 doses were given at 3mg/kgbw.

Example 4: Efficacy Evaluation of Two Doses of Pazopanib Composition inMouse Coronavirus Induced Lung Injury Model 1. Purpose

Evaluate the efficacy of pazopanib composition in coronavirus (MurineHepatitis Virus Strain 1, MHV-1) induced mouse lung injury model.

2. Procedure

a. Pazopanib Composition Preparation

-   -   1. Put 3.294 ml ddH2O in a small beaker with a small magnetic        stir bar.    -   2. Weigh 1.663 g 2-Hydroxypropyl-beta-cyclodextrin (HP-b-CD).    -   3. Add HP-b-CD slowly into the water while stirring.    -   4. Keep stirring until HP-b-CD completely dissolved.    -   5. Transfer the HP-b-CD solution into a 15 ml tube.    -   6. Weigh 43 mg pazopanib hydrochloride and add it into HP-b-CD        solution.    -   7. Vibrate the tube for 5 min followed by 30 min water bath        sonication.    -   8. Then keep the tube in 50° C. for 30min and pazopanib        hydrochloride should be completely dissolved.    -   Vehicle Stock: 33.26% (332.6 mg/ml) HP-b-CD in ddH2O    -   Stock solution of pazopanib: 8.6 mg/ml in vehicle control

Vehicle Stock Pazopanib stock Vehicle control   100 ul   0 ul 3 mg/kgbw93.03 ul 6.97 ul * Each mouse is around 20 gbw and retro-orbitalinjection volume is 100 ul

b. Animal Procedure

-   -   A/J mice, 8-10 weeks old, were anaesthetized by        Ketamine/Xylazine (100 mg/kg and 10 mg/kg) and were kept under        anesthesia during the whole procedure using Ketamine/Xylazine.        After being deeply anesthetized (assessed by applying a noxious        stimulus, eg toe pinch, and observing no reflex response and no        change in either the rate or character of respiration), mice        received an intranasal inoculation of 6000 PFU MHV-1 in 20 μl        Dulbecco's modified Eagle's medium. After the administration,        the mice were monitored until their breathing gradually returned        to normal. Then the mice were returned to the recovery cage on        the heating pad and monitored for their anesthesia status.        Twenty-four hours after the virus inoculation, vehicle control        or 3 mg/kgbw pazopanib was delivered to mice via retro-orbital.        Thirty-three hours after the virus inoculation, vehicle control        or 3 mg/kgbw pazopanib was delivered to mice via retro-orbital        again. Sixteen hours after the 2^(nd) dose of the drug        administration, 100 μl of FITC-labeled albumin (10 mg/ml) was        injected via the retro-orbital vein. Two hours after FITC-album        in injection, mice were euthanized and bronchoalveolar lavage        (BAL) was collected via instilling 1 ml of PBS into the lungs,        which was retrieved via a tracheal catheter. The green        fluorescent of BAL was measured by the plate reader. BAL        fluorescent intensities from pazopanib treated mice were        normalized to the intensities from vehicle control-treated mice.

Conclusion

As shown in FIG. 2, treatment with a pharmaceutical composition ofpazopanib can significantly reduce pulmonary permeability in mousecoronavirus induced acute lung injury when 2 doses were given at 3mg/kgbw.

Example 5: Efficacy Evaluation of One Dose of Pazopanib CompositionGiven 24 Hours After Virus Inoculation in Mouse Coronavirus Induced LungInjury Model Purpose

Evaluate the efficacy of pazopanib composition in coronavirus (MurineHepatitis Virus Strain 1, MHV-1) induced mouse lung injury model.

Procedure

a. Pazopanib Composition Preparation

-   -   1. Put 3.294 ml ddH2O in a small beaker with a small magnetic        stir bar.    -   2. Weigh 1.663 g 2-Hydroxypropyl-beta-cyclodextrin (HP-b-CD).    -   3. Add HP-b-CD slowly into the water while stirring.    -   4. Keep stirring until HP-b-CD completely dissolved.    -   5. Transfer the HP-b-CD solution into a 15 ml tube.    -   6. Weigh 43 mg pazopanib hydrochloride and add it into HP-b-CD        solution.    -   7. Vibrate the tube for 5 min followed by 30 min water bath        sonication.    -   8. Then keep the tube in 50° C. for 30 min and pazopanib        hydrochloride should be completely dissolved.    -   Vehicle Stock: 33.26% (332.6 mg/ml) HP-b-CD in ddH2O    -   Stock solution of pazopanib: 8.6 mg/ml in vehicle control

Vehicle Stock Pazopanib stock Vehicle control   100 ul   0 ul 3 mg/kgbw93.03 ul 6.97 ul * Each mouse is around 20 gbw and retro-orbitalinjection volume is 100 ul

b. Animal Procedure

-   -   A/J mice, 8-10 weeks old, were anaesthetized by        Ketamine/Xylazine (100 mg/kg and 10 mg/kg) and were kept under        anesthesia during the whole procedure using Ketamine/Xylazine.        After being deeply anesthetized (assessed by applying a noxious        stimulus, eg toe pinch, and observing no reflex response and no        change in either the rate or character of respiration), mice        received an intranasal inoculation of 6000 PFU MHV-1 in 20 μl        Dulbecco's modified Eagle's medium. After the administration,        the mice were monitored until their breathing gradually returned        to normal. Then the mice were returned to the recovery cage on        the heating pad and monitored for their anesthesia status.        Twenty-four hours after the virus inoculation, vehicle control        or 3 mg/kgbw pazopanib was delivered to mice via retro-orbital.        Twenty-four hours after the drug administration, 100 μl of        FITC-labeled albumin (10 mg/ml) was injected via the        retro-orbital vein. Two hours after FITC-albumin injection, mice        were euthanized and bronchoalveolar lavage (BAL) was collected        via instilling 1 ml of PBS into the lungs, which was retrieved        via a tracheal catheter. The green fluorescent of BAL was        measured by the plate reader. BAL fluorescent intensities from        pazopanib treated mice were normalized to the intensities from        vehicle control-treated mice.

Conclusion

As shown in FIG. 3, treatment with a pharmaceutical composition ofpazopanib can significantly reduce pulmonary permeability in mousecoronavirus induced acute lung injury model when one dose (3 mg/kgbw)was given at 24 hr after virus inoculation.

Example 6: Efficacy Evaluation of One Dose of Pazopanib CompositionGiven 48 Hours After Virus Inoculation in Mouse Coronavirus Induced LungInjury Model Purpose

Evaluate the efficacy of pazopanib composition in coronavirus (MurineHepatitis Virus Strain 1, MHV-1) induced mouse lung injury model.

Procedure

a. Pazopanib Composition Preparation

-   -   1. Put 3.294 ml ddH2O in a small beaker with a small magnetic        stir bar.    -   2. Weigh 1.663 g 2-Hydroxypropyl-beta-cyclodextrin (HP-b-CD).    -   3. Add HP-b-CD slowly into the water while stirring.    -   4. Keep stirring until HP-b-CD completely dissolved.    -   5. Transfer the HP-b-CD solution into a 15 ml tube.    -   6. Weigh 43 mg pazopanib hydrochloride and add it into HP-b-CD        solution.    -   7. Vibrate the tube for 5 min followed by 30 min water bath        sonication.    -   8. Then keep the tube in 50° C. for 30 min and pazopanib        hydrochloride should be completely dissolved.    -   Vehicle Stock: 33.26% (332.6 mg/ml) HP-b-CD in ddH2O    -   Stock solution of pazopanib: 8.6 mg/ml in vehicle control

Vehicle Stock Pazopanib stock Vehicle control   100 ul   0 ul 3 mg/kgbw93.03 ul 6.97 ul * Each mouse is around 20 gbw and retro-orbitalinjection volume is 100 ul

b. Animal Procedure

-   -   A/J mice, 8-10 weeks old, were anaesthetized by        Ketamine/Xylazine (100 mg/kg and 10 mg/kg) and were kept under        anesthesia during the whole procedure using Ketamine/Xylazine.        After being deeply anesthetized (assessed by applying a noxious        stimulus, eg toe pinch, and observing no reflex response and no        change in either the rate or character of respiration), mice        received an intranasal inoculation of 6000 PFU MHV-1 in 20 μl        Dulbecco's modified Eagle's medium. After the administration,        the mice were monitored until their breathing gradually returned        to normal. Then the mice were returned to the recovery cage on        the heating pad and monitored for their anesthesia status.        Forty-eight hours after the virus inoculation, vehicle control        or 3 mg/kgbw pazopanib was delivered to mice via retro-orbital.        Twenty-four hours after the drug administration, 100 μl of        FITC-labeled albumin (10 mg/ml) was injected via the        retro-orbital vein. Two hours after FITC-albumin injection, mice        were euthanized and bronchoalveolar lavage (BAL) was collected        via instilling 1 ml of PBS into the lungs, which was retrieved        via a tracheal catheter. The green fluorescent of BAL was        measured by the plate reader. BAL fluorescent intensities from        pazopanib treated mice were normalized to the intensities from        vehicle control-treated mice.

Conclusion

As shown in FIG. 4, treatment with a pharmaceutical composition ofpazopanib can significantly reduce pulmonary permeability in mousecoronavirus induced acute lung injury model when one dose (3 mg/kgbw)was given at 48 hr after virus inoculation.

Example 7: Efficacy Evaluation of One Dose of Pazopanib CompositionGiven 72 Hours After Virus Inoculation in Mouse Coronavirus Induced LungInjury Model Purpose

Evaluate the efficacy of pazopanib composition in coronavirus (MurineHepatitis Virus Strain 1,MHV-1) induced mouse lung injury model.

Procedure

a. Pazopanib Composition Preparation

-   -   1. Put 3.294 ml ddH2O in a small beaker with a small magnetic        stir bar.    -   2. Weigh 1.663 g 2-Hydroxypropyl-beta-cyclodextrin (HP-b-CD).    -   3. Add HP-b-CD slowly into the water while stirring.    -   4. Keep stirring until HP-b-CD completely dissolved.    -   5. Transfer the HP-b-CD solution into a 15 ml tube.    -   6. Weigh 43 mg pazopanib hydrochloride and add it into HP-b-CD        solution.    -   7. Vibrate the tube for 5 min followed by 30 min water bath        sonication.    -   8. Then keep the tube in 50° C. for 30 min and pazopanib        hydrochloride should be completely dissolved.    -   Vehicle Stock: 33.26% (332.6 mg/ml) HP-b-CD in ddH2O    -   Stock solution of pazopanib: 8.6 mg/ml in vehicle control

Vehicle Stock Pazopanib stock Vehicle control   100 ul   0 ul 3 mg/kgbw93.03 ul 6.97 ul * Each mouse is around 20 gbw and retro-orbitalinjection volume is 100 ul

b. Animal Procedure

-   -   A/J mice, 8-10 weeks old, were anaesthetized by        Ketamine/Xylazine (100 mg/kg and 10 mg/kg) and were kept under        anesthesia during the whole procedure using Ketamine/Xylazine.        After being deeply anesthetized (assessed by applying a noxious        stimulus, eg toe pinch, and observing no reflex response and no        change in either the rate or character of respiration), mice        received an intranasal inoculation of 6000 PFU MHV-1 in 20 μl        Dulbecco's modified Eagle's medium. After the administration,        the mice were monitored until their breathing gradually returned        to normal. Then the mice were returned to the recovery cage on        the heating pad and monitored for their anesthesia status.        Seventy-two hours after the virus inoculation, vehicle control        or 3 mg/kgbw pazopanib was delivered to mice via retro-orbital.        Twenty-four hours after the drug administration, 100 μl of        FITC-labeled albumin (10 mg/ml) was injected via the        retro-orbital vein. Two hours after FITC-albumin injection, mice        were euthanized and bronchoalveolar lavage (BAL) was collected        via instilling 1 ml of PBS into the lungs, which was retrieved        via a tracheal catheter. The green fluorescent of BAL was        measured by the plate reader. BAL fluorescent intensities from        pazopanib treated mice were normalized to the intensities from        vehicle control-treated mice.

Conclusion

As shown in FIG. 5, treatment with a pharmaceutical composition ofpazopanib has a negligible effect on pulmonary permeability in mousecoronavirus induced acute lung injury model when one dose (3 mg/kgbw)was given at 72 hr after virus inoculation.

Example 8: Efficacy Evaluation of Pazopanib Composition in Body WeightLoss and Survival Probability of Mouse Coronavirus Induced Lung InjuryModel Purpose

Evaluate the efficacy of pazopanib composition in coronavirus (MurineHepatitis Virus Strain 1, MHV-1) induced mouse lung injury model.

Procedure

a. Pazopanib Composition Preparation

-   -   1. Put 3.294 ml ddH2O in a small beaker with a small magnetic        stir bar.    -   2. Weigh 1.663 g 2-Hydroxypropyl-beta-cyclodextrin (HP-b-CD).    -   3. Add HP-b-CD slowly into the water while stirring.    -   4. Keep stirring until HP-b-CD completely dissolved.    -   5. Transfer the HP-b-CD solution into a 15 ml tube.    -   6. Weigh 43 mg pazopanib hydrochloride and add it into HP-b-CD        solution.    -   7. Vibrate the tube for 5 min followed by 30 min water bath        sonication.    -   8. Then keep the tube in 50° C. for 30 min and pazopanib        hydrochloride should be completely dissolved.    -   Vehicle Stock: 33.26% (332.6 mg/ml) HP-b-CD in ddH2O    -   Stock solution of pazopanib: 8.6 mg/ml in vehicle control

Vehicle Stock Pazopanib stock Vehicle control   100 ul   0 ul 3 mg/kgbw93.03 ul 6.97 ul * Each mouse is around 20 gbw and retro-orbitalinjection volume is 100 ul

b. Animal Procedure

-   -   A/J mice, 8-10 weeks old, were anaesthetized by        Ketamine/Xylazine (100 mg/kg and 10 mg/kg) and were kept under        anesthesia during the whole procedure using Ketamine/Xylazine.        After being deeply anesthetized (assessed by applying a noxious        stimulus, eg toe pinch, and observing no reflex response and no        change in either the rate or character of respiration), mice        received an intranasal inoculation of 6000 PFU MHV-1 in 20 μl        Dulbecco's modified Eagle's medium. After the administration,        the mice were monitored until their breathing gradually returned        to normal. Then the mice were returned to the recovery cage on        the heating pad and monitored for their anesthesia status.        Vehicle control or 3 mg/kgbw pazopanib was delivered to mice via        retro-orbital once daily since 24 hr after the virus        inoculation. The last dose was given on 120 hr after the virus        inoculation. The mice were monitored twice daily. Survival and        body weight were recorded.

Conclusion

As shown in FIGS. 6 and 7, treatment with a pharmaceutical compositionof pazopanib does not affect survival rate or body weight loss in mousecoronavirus induced acute lung injury model when one dose was givendaily for 5 days since 24 hr after virus inoculation. These results arenot contrary to or unsupportive of the results in Examples 3-6 thatsufficiently early intervention with a pazopanib composition reducesviral-induced lung injury as quantified by pulmonary permeability.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents, includingcertificates of correction, patent application documents, scientificarticles, governmental reports, websites, and other references referredto herein is incorporated by reference herein in its entirety for allpurposes. In case of a conflict in terminology, the presentspecification controls.

EQUIVALENTS

The invention can be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are to be considered in all respects illustrative ratherthan limiting on the invention described herein. In the variousembodiments of the methods and compositions of the present invention,where the term comprises is used with respect to the recited steps ofthe methods or components of the compositions, it is also contemplatedthat the methods and compositions consist essentially of, or consist of,the recited steps or components. Furthermore, it should be understoodthat the order of steps or order for performing certain actions isimmaterial so long as the invention remains operable. Moreover, two ormore steps or actions can be conducted simultaneously.

In the specification, the singular forms also include the plural forms,unless the context clearly dictates otherwise. Unless defined otherwise,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. In the case of conflict, the present specificationwill control.

Furthermore, it should be recognized that in certain instances acomposition can be described as being composed of the components priorto mixing, because upon mixing certain components can further react orbe transformed into additional materials.

All percentages and ratios used herein, unless otherwise indicated, areby weight. It is recognized the mass of an object is often referred toas its weight in everyday usage and for most common scientific purposes,but that mass technically refers to the amount of matter of an object,whereas weight refers to the force experienced by an object due togravity. Also, in common usage the “weight” (mass) of an object is whatone determines when one “weighs” (masses) an object on a scale orbalance.

What is claimed is:
 1. A method for treating, preventing, or reducingthe severity of a lung injury associated with a coronavirus infectioncomprising administering to a human subject in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising a compound capable of inhibiting MAP3K2/MAP3K3, or apharmaceutically acceptable salt, ester, solvate, or prodrug thereof. 2.The method of claim 1 wherein the coronavirus infection is caused by theSevere Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2).
 3. Themethod of claim 1 wherein said compound capable of inhibitingMAP3K2/MAP3K3 is selected from the group consisting of pazopanib ornintedanib, and combinations thereof, and pharmaceutically acceptablesalts, solvates, or prodrugs thereof.
 4. The method of claim 3 whereinsaid compound capable of inhibiting MAP3K2/MAP3K3 is pazopanib, or apharmaceutically acceptable salt, solvate, or prodrug thereof.
 5. Themethod of claim 4 wherein the pharmaceutically acceptable salt isselected from a salt of a mineral acid selected from the groupconsisting of hydrochloride, hydrobromide, hydroiodide, hydrogensulfate, or a salt selected from mesylate, esylate, besylate, tosylate,and combinations thereof.
 6. The method of claim 5 wherein the compoundcapable of inhibiting MAP3K2/MAP3K3 is pazopanib hydrochloride.
 7. Themethod of claim 3 wherein said compound capable of inhibitingMAP3K2/MAP3K3 is nintedanib, or a pharmaceutically acceptable salt,solvate, or prodrug thereof.
 8. The method of claim 7 wherein thepharmaceutically acceptable salt is selected from a salt of a mineralacid selected from the group consisting of hydrochloride, hydrobromide,hydroiodide, hydrogen sulfate, or a salt selected from mesylate,esylate, besylate, tosylate, and combinations thereof.
 9. The method ofclaim 8 wherein the compound capable of inhibiting MAP3K2/MAP3K3 isnintedanib esylate.
 10. The method of claim 1 wherein the compoundcapable of inhibiting MAP3K2/MAP3K3 is administered using anadministration route selected from the group consisting of oral, intravenous, parenteral, nasal, inhalation (i.e. respiratory), intratracheal,intrapulmonary, and intrabronchial.
 11. The method of claim 10 whereinthe administration is performed using a spray device or nebulizer. 12.The method of claim 1 wherein the lung injury is lung fibrosis.
 13. Themethod of claim 1 wherein the lung injury is selected from the groupconsisting of acute lung injury (ALI) and acute respiratory distresssyndrome (ARDS).
 14. The method of claim 13 wherein the lung injury isacute lung injury (ALI).
 15. The method of claim 13 wherein the lunginjury is acute respiratory distress syndrome (ARDS).
 16. The method ofclaim 1 wherein the lung involves pulmonary edema, and wherein thepulmonary edema is reduced after treatment.
 17. The method of claim 1wherein the patient experiences one or more improvements of conditionafter treatment selected from the group consisting of: reduced time onventilator, improved blood oxygen levels, an increase in rheumatoidfactor (RF) production from neutrophils, an increase in reactive oxygenspecies (ROS), reduced recovery time, reduced pulmonary permeability,improved pulmonary barrier function, increased pulmonary barrier cellsurvival.
 18. The method of claim 1 wherein the composition isadministered from the group consisting of about four times per day,about three times per day, about two times per day, about one time perday, about one time every other day, about two times per week, and aboutone time per week.
 19. The method of claim 18 wherein the composition isadministered for a period selected from the group consisting of about 1day to about 30 days [or about one month], about 1 day to about 14 days,about 1 day to about 10 days, about 1 day to about 1 week (7 days),about 1 day to about 5 days, about 1 day to about 3 days, and until theinfection is treated.
 20. The method of claim 19 wherein at least one ofthe following pharmacokinetic parameters achieved in the patient isselected from an AUC of about 1,037 mcg*h/mL or a Cmax of about 58.1mcg/mL (equivalent to about 132 μM) for the compound capable ofinhibiting MAP3K2/MAP3K3.
 21. The method of claim 1 wherein thecomposition comprises from about 1 to about 1000 mg per unit dosage ofthe compound capable of inhibiting MAP3K2/MAP3K3 based on the activemoiety of the compound.
 22. The method of claim 21 wherein thecomposition comprises about 100 mg, or about 200 mg, or about 300 mg, orabout 400 mg, or about 500 mg or about 600 mg, or about 700 mg, or about800 mg per unit dosage of the compound capable of inhibitingMAP3K2/MAP3K3 based on the active moiety of the compound.
 23. A methodfor treating, preventing, or reducing the severity of a lung injuryassociated with a coronavirus infection comprising administering to ahuman subject in need thereof a therapeutically effective amount of apharmaceutical composition comprising: a compound capable of inhibitingMAP3K2/MAP3K3; and an additional active agent.
 24. The method of claim23 wherein the coronavirus infection is caused by the Severe AcuteRespiratory Syndrome Corona Virus 2 (SARS-CoV-2).
 25. The method ofclaim 23, wherein the compound capable of inhibiting MAP3K2/MAP3K3 isselected from the group consisting of pazopanib or nintedanib, andcombinations thereof, or a pharmaceutically acceptable salt, solvate, orprodrug thereof.
 26. The method of claim 23 wherein the additional agentis remdesivir, or a pharmaceutically acceptable salt, ester, solvate, orprodrug thereof.
 27. The method of claim 1 wherein the lung injury isselected from the group consisting of acute lung injury (ALI), lungfibrosis, and acute respiratory distress syndrome (ARDS).
 28. Apharmaceutical composition for treating, preventing, or reducing theseverity of a lung injury associated with a SARS-CoV-2 infection,comprising: (a) a therapeutically effective amount of a compound capableof inhibiting MAP3K2/MAP3K3, or a pharmaceutically acceptable salt,ester, solvate, or prodrug thereof, and (b) a pharmaceuticallyacceptable carrier.
 29. The pharmaceutical composition of claim 28wherein the compound capable of inhibiting MAP3K2/MAP3K3 is selectedfrom the group consisting of pazopanib or nintedanib, and combinationsthereof, or a pharmaceutically acceptable salt, solvate, or prodrugthereof.
 30. The pharmaceutical composition of any of claim 28 furthercomprising an additional active agent, wherein the additional agent isremdesivir, or a pharmaceutically acceptable salt, ester, solvate, orprodrug thereof.